Studies of a novel 3-0 sulfatase specific for a unique glucosamine residue associated with a short antithrombin binding sequence of heparin have been extended. The enzyme has been demonstrated in human and rat urine and in plasma, but not in dialyzed extracts of rat liver, kidney, spleen, lung and testis. The enzyme may arise from circulating erythrocytes or platelets. However, not all tissues have been examined -- intestine, for example, and the possibility that non-dialysable heparin like mucopolysaccharides may mask the presence of the enzyme has not been excluded. The synthesis of O-sulfated alpha-methyl glucosaminide derivatives of known structure has required the synthesis of nine intermediates. Each of these has now been obtained as pure crystalline compounds and some of their physical properties determined. In collaboration with Di Natale, the diagnosis of the Sanfilippo-C syndrome in extracts of leukocytes was carried out using glucosamine and radioactively labeled acetyl-CoA as substrates. In related experiments, it was found that alpha-methyl N-acetyl glucosaminide 6-0 sulfate could not replace more complex sugars as a substrate for the 6-0 sulfatase associated with the Sanfilippo-D syndrome.